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Macrophage elastase [matrix metalloproteinase (MMP)-12] is the most upregulated MMP in abdominal aortic aneurysm (AAA) and, hence, MMP-12-targeted imaging may predict AAA progression and rupture risk. Here, we report the design, synthesis, and evaluation of three novel hydroxamate-based selective MMP-12 inhibitors (CGA, CGA-1, and AGA) and the methodology to obtain MMP-12 selectivity from hydroxamate-based panMMP inhibitors. Also, we report two 99m Tc-radiotracers, 99m Tc-AGA-1 and 99m Tc-AGA-2, derived from AGA. 99m Tc-AGA-2 displayed faster blood clearance in mice and better radiochemical stability compared to 99m Tc-AGA-1. Based on this, 99m Tc-AGA-2 was chosen as the lead tracer and tested in murine AAA. 99m Tc-AGA-2 uptake detected by autoradiography was significantly higher in AAA compared to normal aortic regions. Specific binding of the tracer to MMP-12 was demonstrated through ex vivo competition. Accordingly, this study introduces a novel family of selective MMP-12 inhibitors and tracers, paving the way for further development of these agents as therapeutic and imaging agents.

A new near-infrared fluorescent compound containing two cyclic RGD motifs, cypate-[c(RGDfK)](2) (1), was synthesized based on a carbocyanine fluorophore bearing two carboxylic acid groups (cypate) for integrin α(v)β(3)-targeting. Compared with its monovalent counterpart cypate-c(RGDfK) (2), 1 exhibited remarkable improvements in integrin α(v)β(3) binding affinity and tumor uptake in nude mice of A549. The results suggest that cypate-linked divalent ligands can serve as an important molecular platform for exploring receptor-targeted optical imaging and treatment of various diseases., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

We synthesized disulfide-based cyclic RGD pentapeptides bearing a near-infrared fluorescent dye (cypate), represented by cypate-c(CRGDC) (1) for integrin-targeted optical imaging. These compounds were compared with the traditional lactam-based cyclic RGD counterpart, cypate-c(RGDfK) (2). Molecular modeling suggests that the binding affinity of 2 to integrin α(v)β(3) is an order of magnitude higher than that of 1. This was confirmed experimentally, which further showed that substitution of Gly with Pro, Val and Tyr in 1 remarkably hampered the α(v)β(3) binding. Interestingly, cell microscopy with A549 cells showed that 1 exhibited higher cellular staining than 2. These results indicate that factors other than receptor binding affinity to α(v)β(3) dimeric proteins mediate cellular uptake. Consequently, 1 and its analogs may serve as valuable molecular probes for investigating the selectivity and specificity of integrin targeting by optical imaging., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Recently, a disulfide-based cyclic RGD peptide called iRGD, that is, c(CRGDKGPDC), has been reported to interact with both integrin and neuropilin-1 receptors for cellular and deep tissue penetration to improve the imaging sensitivity and therapeutic efficacy. In this study, two new near-infrared fluorescent iRGD conjugates, that is, Ac-Cys(IRDye®800CW)-iRGD (1), and its dual labeling analog DOTA-Cys(IRDye®800CW)-iRGD (2) were synthesized via the specific mercapto-maleimide reaction for tumor imaging. Both 1 and 2 showed significant tumor localization in optical imaging of MDA-MB-435 tumor-bearing mice. The potential of such iRGD compounds in tumor-targeted imaging and drug delivery deserves further exploration., (Published by Elsevier Ltd.)

Objectives: The alpha(v)beta(3) integrin is a cell adhesion molecule known to be involved in stages of angiogenesis and metastasis. In this study, the chelators CB-TE2A and diamsar were conjugated to cyclic RGDyK and RGDfD and the biological properties of (64)Cu-labeled peptides were compared., Methods: CB-TE2A-c(RGDyK) and diamsar-c(RGDfD) were labeled with (64)Cu in 0.1 M NH(4)OAc (pH=8) at 95 degrees C and 25 degrees C, respectively. PET and biodistribution studies were carried out on M21 (alpha(v)beta(3)-positive) and M21L (alpha(v)-negative) melanoma-bearing mice. Binding affinity of the Cu-chelator-RGD peptides to alpha(v)beta(3) integrins was determined by a competitive binding affinity assay., Results: Biological studies showed higher concentration of (64)Cu-CB-TE2A-c(RGDyK) in M21 tumor compared to M21L tumor at 1 and 4 h pi. Tumor concentration of (64)Cu-CB-TE2A-c(RGDyK) was higher than that of (64)Cu-diamsar-c(RGDfD). The difference is not due to differing binding affinities, since similar values were obtained for the agents. Compared to (64)Cu-diamsar-c(RGDfD), there is more rapid liver and blood clearance of (64)Cu-CB-TE2A-c(RGDyK), resulting in a lower liver and blood concentration at 24 h pi. Both (64)Cu-labeled RGD peptides show similar binding affinities to alpha(v)beta(3). The differences in their biodistribution properties are likely related to different linkers, charges and lipophilicities. The M21 tumor is clearly visualized with (64)Cu-CB-TE2A-c(RGDyK) by microPET imaging. Administration of c(RGDyK) as a block significantly reduced the tumor concentration; however, the radioactivity background was also decreased by the blocking dose., Conclusions: Both (64)Cu-CB-TE2A-c(RGDyK) and (64)Cu-diamsar-c(RGDfD) are potential candidates for imaging tumor angiogenesis. For diamsar-c(RGDfD), a linker may be needed between the Cu-chelator moiety and the RGD peptide to achieve optimal in vivo tumor concentration and clearance from nontarget organs.

Phosphinic compounds have potential as amide-bond mimetics in the development of novel peptidomimetics, enzyme inhibitors, and metal-binding ligands. Novel pseudo-oligopeptides with two phosphinic acid groups embedded in the peptide backbone serving as amide-bond surrogates, Psi[P(O,OH)--CH(2)], were targeted. A series of linear and cyclic pseudo-oligopeptides with two phosphinic acid groups arrayed at different positions in the peptide sequence were designed, including Ac--Phe--{(R,S)--AlaPsi[P(O,OH)--CH(2)]Gly}(2)--NH(2) (P2), Ac--NH--(R,S)--AlaPsi[P(O,OH)--CH(2)]Gly--Phe--(R,S)--AlaPsi[P(O,OH)--CH(2)]Gly--NH(2) (P3), Ac--NH--(R,S)--AlaPsi[P(O,OH)--CH(2)]Gly--Phe--Phe--(R,S) --AlaPsi[P(O,OH)--CH(2)]Gly--NH(2) (P4), cyclo{NH--(R,S)--AlaPsi[P(O,OH)--CH(2)]Gly--Phe}(2) (P5), and cyclo[NH--(R,S)--AlaPsi[P(O,OH)--CH(2)]Gly--Phe--Phe](2) (P6). They were synthesized via conventional Fmoc chemistry on solid support utilizing Fmoc-protected phosphinic acid-containing pseudo-dipeptide fragment, i.e. Fmoc--(R,S)--AlaPsi[P(O,OCH(3))--CH(2)]Gly--OH. The pseudo-peptides containing two phosphinic acid groups exhibited the highest binding affinity and selectivity for Fe(III) among the 10-metal ions screened by ESI-MS analysis--Cu(II), Zn(II), Co(II), Ni(II), Mn(II), Fe(II), Fe(III), Al(III), Ga(III), and Gd(III). P4 and P6 with 11-atom linkages between the two phosphinic acids preferred intramolecular metal binding to form 1:1 ligand/metal complexes. As revealed by competition experiments, P4 showed the highest relative binding affinity among the six compounds tested. Noteworthy, P4 also showed higher relative binding affinity than similar dihydroxamate-containing pseudo-peptides reported previously. The novel structural prototype and facile synthesis along with selective and potent Fe(III) binding strongly suggest that pseudo-peptides containing the two or more phosphinic groups as amide-bond surrogates deserve further exploration in medicinal chemistry.

Unlabelled: Bone diseases are often a result of increased numbers of osteoclasts, or bone-resorbing cells. Bone metastases are a significant cause of morbidity in many types of cancer. An imaging agent targeting osteoclasts, which are upregulated in osteolytic lesions, may facilitate earlier follow-up in patients with osteolytic or mixed bone metastases. Osteoclasts express high levels of alpha(v)beta3 integrin, to which peptides containing the Arg-Gly-Asp (RGD) sequence are known to bind. We proposed that radiolabeled RGD peptides could be used to detect osteoclasts in lytic bone lesions., Methods: The cross-bridged macrocyclic chelator 4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane (CB-TE2A) was conjugated to c(RGDyK) for radiolabeling with 64Cu (t(1/2), 12.7 h; beta+, 17.4%; E(beta+ max), 656 keV; beta-, 39%; E(beta- max), 573 keV). The in vitro affinity of Cu(II)-CB-TE2A-c(RGDyK) for alpha(v)beta3 and alpha(v)beta5 was evaluated in a heterologous competitive binding assay. Ex vivo uptake was examined in osteoclasts prepared from bone marrow macrophages. As a proof of principle, biodistribution and imaging studies were performed on parathyroid hormone (PTH)-induced osteolysis in the calvarium., Results: Cu-CB-TE2A-c(RGDyK) was shown to have a 30-fold higher affinity for alpha(v)beta3 than for alpha(v)beta5. Osteoclasts were shown to specifically take up (64)Cu-CB-TE2A-c(RGDyK). However, bone marrow macrophages showed only nonspecific uptake. PTH treatment increased calvarial uptake of 64Cu-CB-TE2A-c(RGDyK), compared with uptake in mice receiving a sham treatment. In addition, calvarial uptake correlated linearly with the number of osteoclasts on the bone surface., Conclusion: These results suggest that 64Cu-CB-TE2A-c(RGDyK) selectively binds alpha(v)beta3 on osteoclasts and may potentially be used to identify increased numbers of osteoclasts in osteolytic bone diseases such as osteolytic bone metastasis and inflammatory osteolysis.

Biomolecules containing the RGD peptide sequence are known to bind integrins with high affinity. Studies of hexa-and hepta-peptides labeled with a near-infrared fluorescent probe (cypate) showed that rearranging the glycine in a linear RGD peptide sequence to form the GRD analogue favored the uptake of the GRD compound by alphavbeta3 integrin receptor (ABIR)-positive A549 tumor cells and tissue. The internalization of the GRD compound in A549 cells and tumor uptake in mice were inhibited by ABIR-avid peptides, suggesting its recognition by this receptor. Further studies with functional blocking antibodies and beta3 knockout cells revealed that beta3 integrin mediates the internalization of the cypate-GRD peptide. Molecular modeling studies supported preferential interaction of the probe with the beta3 subunit of integrins relative to the alphav subunit. The results demonstrate that the cypate-GRD peptide targets beta3 integrin, thereby providing a strategy to monitor drug delivery and efficacy, and physiopathologic processes mediated by this protein.

Molecular interactions between RGD peptides and integrins are known to mediate many biological and pathological processes. This has led to an increased interest in the development of RGD compounds with high affinity and improved selectivity for integrin receptors. In this study, we synthesized and evaluated a series of multimeric RGD compounds constructed on a dicarboxylic acid-containing near-infrared (NIR) fluorescent dye (cypate) for tumor targeting. An array of NIR fluorescent RGD compounds was prepared efficiently, including one RGD monomer (cypate-(RGD)(2)-NH(2)), two RGD dimers (cypate-(RGD)(2)-NH(2) and cypate-(RGD-NH(2))(2)), one trimer (cypate-(RGD)(3)-NH(2)), two tetramers (cypate-(RGD)(4)-NH(2) and cypate-[(RGD)(2)-NH(2)](2)), one hexamer (cypate-[(RGD)(3)-NH(2)](2)), and one octamer (cypate-[(RGD)(4)-NH(2)](2)). The binding affinity of the multimeric RGD compounds for alpha(v)beta(3) integrin receptor (ABIR) showed a remarkable increase relative to the monomer cypate-RGD-NH(2). Generally, the divalent linear arrays of the multimeric RGD units bound the ABIR with slightly higher affinity than their monovalent analogues. These results suggest that the receptor binding affinity was not only dependent on the number of RGD moieties but also on the spatial alignments of the pendant peptides. Internalization of the compounds by ABIR-positive tumor cells (A549) was monitored by NIR fluorescence microscopy. The data showed that endocytosis of the octameric RGD derivative was significantly higher by comparison to other compounds in this study. In vivo noninvasive optical imaging and biodistribution data showed that the compounds were retained in A549 tumor tissue. These results clearly demonstrated that an array of simple RGD tripeptides on a NIR fluorescent dye core can be recognized by ABIR. Optimization of the spatial alignment of the RGD moieties through careful molecular design and library construction could induce multivalent ligand-receptor interactions useful for in vivo tumor imaging and tumor-targeted therapy.

Integrins mediate many biological processes, including tumor-induced angiogenesis and metastasis. The arginine-glycine-aspartic acid (RGD) peptide sequence is a common recognition motif by integrins in many proteins and small peptides. While evaluating a small library of RGD peptides for imaging alpha(V)beta(3) integrin (ABI)-positive tumor cell line (A549) by optical methods, we discovered that conjugating a presumably inactive linear hexapeptide GRDSPK with a near-infrared carbocyanine molecular probe (Cypate) yielded a previously undescribed bioactive ligand (Cyp-GRD) that targets ABI-positive tumors. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay with A549 cells showed that Cyp-GRD was not cytotoxic up to 100 muM in cell culture. The compound was internalized by cells, and this internalization was blocked by coincubation with a cyclic RGD peptide (cyclo[RGDfV], f is d-phenylalanine) that binds ABI with high affinity. In vivo, Cyp-GRD selectively accumulated in tumors relative to surrounding normal tissues. Blocking studies with cyclo[RGDfV] inhibited the in vivo uptake of Cyp-GRD, suggesting that both compounds target the same active site of the protein. A strong correlation between the Cyp-GRD peptide and mitochondrial NADH concentration suggests that the new molecule could also report on the metabolic status of cells ex vivo. Interestingly, neither a Cypate-labeled linear RGD peptide nor an (111)In-labeled DOTA-GRD conjugate was selectively retained in the tumor. These results clearly demonstrate the synergistic effects of Cypate and GRD peptide for molecular recognition of integrin expression and suggest the potential of using carbocyanines as optical scaffolds for designing biologically active molecules.

Macrocyclization of a bioactive peptide with a bifunctional near-infrared fluorescent optical probe gives a compound that retained the receptor binding affinity of the peptide and the photophysical properties of the optical probe. The robust nature of the new compound provides a structural framework for optimizing the activity of bioactive molecules and for monitoring chemical or biological processes in vivo and in vitro by near-infrared optical methods.